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Diabetologia

, Volume 62, Issue 10, pp 1901–1914 | Cite as

Differential expression and release of exosomal miRNAs by human islets under inflammatory and hypoxic stress

  • Prathab Balaji Saravanan
  • Srividya Vasu
  • Gumpei Yoshimatsu
  • Carly M. Darden
  • Xuan Wang
  • Jinghua Gu
  • Michael C. LawrenceEmail author
  • Bashoo NaziruddinEmail author
Article

Abstract

Aims/hypothesis

Pancreatic islets produce non-coding microRNAs (miRNAs) that regulate islet cell function and survival. Our earlier investigations revealed that human islets undergo significant damage due to various types of stresses following transplantation and release miRNAs. Here, we sought to identify and validate exosomal miRNAs (exo-miRNAs) produced by human islets under conditions of cellular stress, preceding loss of cell function and death. We also aimed to identify islet stress signalling pathways targeted by exo-miRNAs to elucidate potential regulatory roles in islet cell stress.

Methods

Human islets were subjected to proinflammatory cytokine and hypoxic cell stress and miRNA from exosomes was isolated for RNA sequencing and analysis. Stress-induced exo-miRNAs were evaluated for kinetics of expression and release by intact islets for up to 48 h exposure to cytokines and hypoxia. A subset of stress-induced exo-miRNAs were assessed for recovery and detection as biomarkers of islet cell stress in a diabetic nude mouse xenotransplant model and in patients undergoing total pancreatectomy with islet auto-transplantation (TPIAT). Genes and signalling pathways targeted by stress-induced exo-miRNAs were identified by Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and direct interactions of miRNAs with downstream signalling targets were validated in human islet cells using the miRNA Tests for Read Analysis and Prediction (MirTrap) system.

Results

Global exo-miRNA sequencing revealed that 879 miRNA species were released from human islets and 190 islet exo-miRNAs were differentially expressed in response to proinflammatory cytokines, hypoxia or both. Release of exo-miRNAs hsa-miR-29b-3p and hsa-miR-216a-5p was detected within 6 h of exposure to cytokines and hypoxia. The remaining subset of stress-induced exo-miRNAs, including hsa-miR-148a-3p and islet cell damage marker hsa-miR-375, showed delayed release at 24–48 h, correlating with apoptosis and cell death. Stress and damage exo-miRNAs were significantly elevated in the circulation in human-to-mouse xenotransplant models and in human transplant recipients. Elevated blood exo-miRNAs negatively correlated with post-transplant islet function based on comparisons of stress and damage exo-miRNA indices with Secretory Unit of Islet Transplant Objects (SUITO) indices. KEGG analysis and further validation of exo-miRNA targets by MirTrap analysis revealed significant enrichment of islet mRNAs involved in phosphoinositide 3-kinase/Akt and mitogen-activated protein kinase signalling pathways.

Conclusions/interpretation

The study identifies exo-miRNAs differentially expressed and released by islets in response to damage and stress. These exo-miRNAs could serve as potential biomarkers for assessing islet damage and predicting outcomes in islet transplantation. Notably, exo-miRNAs 29b-3p and 216a-5p could be detected in islets prior to damage-released miRNAs and indicators of cellular apoptosis and death. Thus, these stress-induced exo-miRNAs may have potential diagnostic value for detecting early islet stress prior to progressive loss of islet cell mass and function. Further investigations are warranted to investigate the utility of these exo-miRNAs as early indicators of islet cell stress during prediabetic conditions.

Keywords

Cellular stress Exosomal miRNA Islet cell damage Islet transplantation PI3K–Akt signalling pathway 

Abbreviations

BAD

BCL2-associated death promoter

Casp-3

Caspase-3

CC

Cytokine cocktail

c-Casp-3

Cleaved Casp-3

CHOP

CCAAT-enhancer-binding protein homologous protein

DMI

Damage miRNA index

D-miR

Damaged-induced exo-miRNA

ER

Endoplasmic reticulum

exo-miRNA

Exosomal miRNA

FDA

Fluorescein diacetate

FOXO1

Forkhead box protein O1

HIF-1α

Hypoxia inducible factor 1α

Hsp70

Heat-shock protein 70

HYP

Hypoxia (experimental treatment)

IEQ

Islet equivalent

IRE

Inositol-requiring enzyme

KEGG

Kyoto Encyclopedia of Genes and Genomes

miRNA

MicroRNA

MirTrap

miRNA Tests for Read Analysis and Prediction

mTOR

Mammalian target of rapamycin

NTA

Nanoparticle tracking analysis

PI

Propidium iodide

PI3K

Phosphoinositide 3-kinase

qPCR

Quantitative PCR

SMI

Stress miRNA index

S-miR

Stress-induced exo-miRNA

STZ

Streptozotocin

SUITO

Secretory Unit of Islet Transplant Objects

TEM

Transmission electron microscopy

TPIAT

Total pancreatectomy with islet auto-transplantation

XBP1

X-box binding protein 1 isoform

Notes

Acknowledgements

Technical support by Y. Liu and A. Rahman, Islet Cell Laboratory, Baylor Simmons Transplant Institute, is acknowledged.

Contribution statement

PBS designed the study, performed experiments, analysed data and wrote the manuscript. SV analysed data and wrote and revised the manuscript. GY and CMD assisted in performing in vivo experiments, data analysis and manuscript preparation. XW and JG analysed microRNA sequencing data and assisted in manuscript preparation. MCL and BN designed and supervised the study, analysed data and wrote the manuscript. All authors approve the final version of the manuscript. BN is responsible for the integrity of the work as a whole.

Funding

The study was supported by an institutional grant from Baylor University Medical Center.

Duality of interest

The authors declare that there is no duality of interest associated with this manuscript.

Supplementary material

125_2019_4950_MOESM1_ESM.pdf (564 kb)
ESM (PDF 563 kb)

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Division of Transplantation, Department of Surgery, Virginia Commonwealth UniversityMedical CenterRichmondUSA
  2. 2.Islet Cell LaboratoryBaylor Scott and White Research InstituteDallasUSA
  3. 3.Islet Cell LaboratoryBaylor Simmons Transplant InstituteDallasUSA

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